Seahorse - Mitochondrial Stress Test: Difference between revisions
Wrote initial page for seahorse assay |
→The Day of the Assay: clarified what is being added to ports |
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[[ Category: Seahorse ]] | [[ Category: Seahorse ]] | ||
[[ Category: Mitochondrial Function ]] | [[ Category: Mitochondrial Function ]] | ||
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==Materials== | ==Materials== | ||
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*** 1 mM Pyruvate (100 uL of a 1M stock) | *** 1 mM Pyruvate (100 uL of a 1M stock) | ||
** Re-adjust the pH to 7.4 (7.35-7.45) | ** Re-adjust the pH to 7.4 (7.35-7.45) | ||
* Wash cells twice with 500 | * Wash cells twice with 500 uL of pre-warmed media, add 500 uL final volume to each well | ||
* Incubate for 1h in non-CO2, humidified, 37C incubator to get rid of any extra CO2 | * Incubate for 1h in non-CO2, humidified, 37C incubator to get rid of any extra CO2 | ||
* While plate is incubating, prepare injection solutions in 1.8 mL of prepared XF Media. These are standard concentrations, and should be optimized for your system (especially FCCP) | * While plate is incubating, prepare injection solutions in 1.8 mL of prepared XF Media. These are standard concentrations, and should be optimized for your system (especially FCCP) | ||
** '''Tube A''' 18 uL Oligomycin | ** '''Tube A''' 18 uL Oligomycin. Will add 56 uL to each port | ||
** '''Tube B''' 18 uL FCCP | ** '''Tube B''' 18 uL FCCP. Will add 62 uL to each port | ||
** '''Tube C''' 9 uL Rotenone and 9 uL Antimycin A | ** '''Tube C''' 9 uL Rotenone and 9 uL Antimycin A. Will add 69 uL to each port | ||
* Add the compounds to the injection ports in the cartridge (A, bottom right; B bottom left; C top right). '''Make sure the barcode is on the right hand side.''' | * Add the compounds to the injection ports in the cartridge (A, bottom right; B bottom left; C top right). '''Make sure the barcode is on the right hand side.''' | ||
* Set up your protocol, editing a similar template and saving with the date and experiment type | * Set up your protocol, editing a similar template and saving with the date and experiment type | ||
* Start the protocol, inserting the cartridge. '''Make sure the barcode is on the right hand side.''' | * Start the protocol, inserting the cartridge with the lid removed. '''Make sure the barcode is on the right hand side.''' | ||
* Once the cartridge is calibrated insert the cell plate at the prompt. If the calibration fails, abort the run and try the calibration again | * Once the cartridge is calibrated insert the cell plate at the prompt. If the calibration fails, abort the run and try the calibration again | ||